The cell surface glycoprotein CD8 serves as a marker for the differentiation of T-cells into the cytotoxic/regulatory cell lineage. In CD8-alpha knockout mice this lineage fails to develop indicating the critical functional role of CD8. The predominant form of CD8 on these T-cells is the CD8 alpha/beta heterodimer. In contrast, a subset of human NK cells and intraepithelial lymphocytes that are TCR gamma/delta exclusively express CD8 alpha/alpha homodimers. Our goal is to determine how the linked CD8-alpha and CD8-beta genes are genetically regulated during T-cell development as a model system for understanding T-cell differentiation. Specifically, we plan to locate regulatory elements such as silencer/enhancers and perform a molecular analysis of the elements to marker gene, we hope to be able to target expression of a marker gene to CD8 expressing cells. Critical regulatory elements will be identified by DNase I hypersensitivity mapping and by testing genomic constructs in vivo using transgenic animals. We plan to either: (i) link regions with hypersensitive sites (HS) to a marker gene; or (ii) delete regions containing HS sites from a human 95 kb fragment that we found allows for expression of the CD8-beta gene in transgenic murine CD8+ T-cells but not CD4+ T-cells. For deleting regions, we will make use of a new yeast/bacterial vector ideally suited for manipulating large pieces of DNA. Expression patterns on thymocytes, mature lymphoid cells, and intraepithelial lymphocytes will be monitored with mAbs and flow cytometry. The identified critical regions will be characterized to identify protein binding sites and to then characterize the binding proteins. Assays proposed include DNase I footprinting, EMSAs, and mutational/functional assays with reporter genes. This work will lead to a better understanding of normal T-cell differentiation which forms a basis for designing strategies to manipulate leukemias and other lymphoid tumors that have abnormal differentiation. We should be able to identify regulatory elements that will allow for the targeting of genes to cytotoxic/suppressor cells. These cells have promise as immunotherapeutic agents by in vitro sensitization and injection into patients. Being able to target genes to this lineage could enhance the ability to develop these cells for immunotherapy.